Part:BBa_K4275027

eforRed-Ag3

EforRed-Ag3 acts as a reporter protein for the verification of the antigen-nanobody interaction between the Ag3 antigen domain and the nanobody domain provided in BBa_K4275026, by showing fluorescent characteristics while fixed onto the nanobody domain.

Figure 1 The 3D structure of the protein predicted by Alphafold2.

Usage and Biology

The reporter protein can be used to test the antigen-nanobody interaction between Ag3 domain and Nb3 domain [1]. by mixing this fusion protein with E.coli with nanobodies presented on the surface. The antigen domain as well as the eforRed domain [2] can be fixed onto the surface of the bacteria. After centrifugation, red fluorescent signal can be observed in the sediment, verifying the cohesion generated by the antigen-nanobody interaction.

eforRED as A Reporter Protein

The nanobody-antigen interaction was verified by mixing intact E.coli cells displaying Neae-Nb3 with the supernatant of Ag3-eforRED (Fig. 2A). Red fluorescent characteristics were observed in the pellets after resuspending the centrifuged mixture, which is absent in the control group that only contains Neae-Nb3 (Fig. 3C).

After that, the type II cohesin-dockerin interaction was tested using the mixture of Neae-Nb3, OlpB-Ag3, and the type II dockerin fused with eforRED (Fig. 2B). A negative control lacking OlpB-Ag3 was set up for result comparison. Centrifugation was used to remove supernatant and the red fluorescence was only identified in pellets of the sample group, confirming the type II cohesin-dockerin interaction (Fig. 3D).

Finally, the association between type I cohesin and type I dockerin was validated using the mixture of Neae-Nb3, OlpB-Ag3, CipA1B2C, and DocI-eforRED (Fig. 2C), red fluorescence was detected in the resuspended mixture while it was not observed in the control group lacking the primary scaffold CipA1B2C (Fig. 3E), verifying the type I cohesin-dockerin interaction.

Figure 2: Cellulosomal scaffold system construct (A) Antigen-nanobody interaction between Nb3 and Ag3 domain reported by the ligated eforRed fluorescent domain (B) Type II cohesin -dockerin interaction between a fixed secondary scaffoldin component and a type II dockerin domain reported by the ligated eforRed fluorescent domain (C) Type I cohesin-dockerin interaction between a fixed primary scaffoldin component and a type I dockerin domain reported by a ligated eforRed fluorescent domain (D) Genetic circuit designed for the expression of ligated form of Ag3, type I dockerin and type II dockerin domains fused with an eforRed fluorescent domain at N terminus for reporting the adhesive functions of those domains.

Figure 3: Production and assay of the scaffold proteins (A) SDS-PAGE analysis for the presence of type I dockerin-eforRed (B) SDS-PAGE analysis of Ag3-eforRed and type II dockerin -eforRed containing type II dockerin fused with an eforRed domain (C) The fluorescence indication for antigen-nanobody interaction for E.coli surface display with an Ag3 control group and a sample group (D) The fluorescence indication for type II cohesin-dockerin interactions with a type II dockerin control group and a sample group (E) The fluorescence indication for type I cohesin-dockerin interactions with a type I dockerin control group and a sample group.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 700
  

References

1. Glass, David S, and Ingmar H Riedel-Kruse. “A Synthetic Bacterial Cell-Cell Adhesion Toolbox for Programming Multicellular Morphologies and Patterns.” Cell vol. 174,3 (2018): 649-658.e16. doi:10.1016/j.cell.2018.06.041

2. Part: "Bba K592012 - Parts.Igem.Org". Parts.Igem.Org, 2022, https://parts.igem.org/Part:BBa_K592012.